Stella modulates transcriptional and endogenous retrovirus programs during maternal-to-zygotic transition Yun Huang1,7 , Jong Kyoung Kim2,6,7, Dang Vinh Do1, Caroline Lee1, Christopher A. Penfold1, Jan J. Zylicz1, John C. Marioni2,3,5, Jamie A. Hackett1,4,8, M. Azim Surani1,8 1 Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK; Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, Cambridge CB2 3DY, UK 2 European Molecular Biology Laboratory – European Bioinformatics Institute (EMBL-EBI), Wellcome Trust Genome Campus, Cambridge CB10 1SD, UK 3 Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Cambridge CB10 1SA, UK 4 Present Address: European Molecular Biology Laboratory (EMBL) - Monterotondo, via Ramarini 32, 00015 Rome, Italy 5 Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge CB2 0RE, UK 6 Department of New Biology, DGIST, Daegu, 42988, Republic of Korea 7 Co-first authors 8 Co-corresponding authors [email protected] [email protected] 1 1 Abstract 2 3 The maternal-to-zygotic transition (MZT) marks the period when the embryonic genome is 4 activated and acquires control of development. Maternally inherited factors play a key role in 5 this critical developmental process, which occurs at the 2-cell stage in mice. We investigated 6 the function of the maternally inherited factor Stella (encoded by Dppa3) using single- 7 cell/embryo approaches. We show that loss of maternal Stella results in widespread 8 transcriptional mis-regulation and a partial failure of MZT. Strikingly, activation of 9 endogenous retroviruses (ERVs) is significantly impaired in Stella maternal/zygotic knockout 10 embryos, which in turn leads to a failure to upregulate chimeric transcripts. Amongst ERVs, 11 MuERV-L activation is particularly affected by the absence of Stella, and direct in vivo 12 knockdown of MuERV-L impacts the developmental potential of the embryo. We propose 13 that Stella is involved in ensuring activation of ERVs, which themselves play a potentially 14 key role during early development, either directly or through influencing embryonic gene 15 expression. 2 16 Introduction 17 18 Maternally inherited factors in the zygote play a critical role during early development 19 (Ancelin et al. 2016, Li et al. 2010, Wasson et al. 2016). The maternal-to-zygotic transition 20 (MZT) marks the time of transfer of developmental control to the embryo following activation 21 of the zygotic genome (Lee et al. 2014, Li et al. 2013). In mice, the major wave of zygotic 22 genome activation (ZGA) occurs at the late 2-cell stage (Golbus et al. 1973). The earliest 23 zygotically transcribed genes are preferentially enriched in essential processes such as 24 transcription, RNA metabolism and ribosome biogenesis (Hamatani et al. 2004, Xue et al. 25 2013). Other events that characterise early pre-implantation development include extensive 26 erasure of global DNA methylation and dynamic changes in histone modifications (Cantone 27 et al. 2013). 28 29 Previous studies have shown that Stella, encoded by the Dppa3 gene locus, is a maternally 30 inherited factor that is required for normal pre-implantation development (Bortvin et al. 2004, 31 Payer et al. 2003). Stella is a small basic protein with a putative SAP-like domain and 32 splicing-like factor; it also contains nuclear localisation and export signal and has the 33 potential to bind to DNA and RNA in-vitro (Nakamura et al. 2007, Payer et al. 2003) (Figure 34 1 – Figure Supplement 1A). Expression of Stella is high in oocytes, continues through pre- 35 implantation development, and subsequently occurs specifically in primordial germ cells. 36 Stella is also expressed in naïve embryonic stem cells, but is downregulated upon exit from 37 pluripotency (Hayashi et al. 2008). Lack of maternal inheritance of Stella results in 38 developmental defects; these manifest from the 2-cell stage onwards, and result in only a 39 small proportion of embryos developing to the blastocyst stage. Notably, a maternally 40 inherited pool of Stella in zygotic Stella knockout embryos, derived from Stella heterozygous 41 females, allows development to progress normally. This indicates Stella has a key role 42 during the earliest developmental events (Payer et al. 2003). 43 3 44 Stella was suggested to protect maternal pronuclei (PN) from TET3 mediated active DNA 45 demethylation (Nakamura et al. 2007, Nakamura et al. 2012) (Figure 1 – Figure Supplement 46 1B). In the absence of Stella, zygotes display enrichment of 5hmC in both parental PN 47 (Wossidlo et al. 2011), and an aberrant accumulation of γH2AX in maternal chromatin 48 (Nakatani et al. 2015). In addition, Stella protects DNA methylation levels at selected 49 imprinted genes and transposable elements (Nakamura et al. 2007). However, embryos 50 depleted of maternal effect proteins known to regulate imprinted genes only exhibit 51 developmental defects post-implantation (Denomme et al. 2013), implying that the 2-cell 52 phenotype in Stella maternal/zygotic knockout (Stella M/Z KO) embryos is not primarily 53 linked with imprint defects. Moreover, TET3 only partially contributes to DNA demethylation 54 and its absence is compatible with embryonic development (Amouroux et al. 2016, Peat et al. 55 2014, Shen et al. 2014, Tsukada et al. 2015). Thus, what impairs pre-implantation 56 embryonic development in the absence of maternal Stella remains unclear. 57 58 A significant number of transposable elements (TEs) are preferentially activated during early 59 development and in a sub-population of mouse embryonic stem cells (Gifford et al. 2013, 60 Macfarlan et al. 2012, Rowe et al. 2011). Notably, at the 2-cell (2C) stage in mouse 61 development, there is selective upregulation of endogenous retrovirus (ERVs), which are a 62 subset of TEs characterised by the presence of LTRs that mediate expression and 63 retrotransposition (Kigami et al. 2003, Ribet et al. 2008). Growing evidence suggests that 64 activation of some TEs has important biological functions during early development (Beraldi 65 et al. 2006, Kigami et al. 2003). TEs can regulate tissue-specific gene expression or splicing 66 through their exaptation as gene regulatory elements, and may also play a key role during 67 speciation (Bohne et al. 2008, Gifford et al. 2013, Rebollo et al. 2012). TEs additionally drive 68 expression of genes directly by acting as alternative promoters that generate chimeric 69 transcripts, which include both TE and protein-coding sequences (Macfarlan et al. 2012, 70 Peaston et al. 2004). Thus the expression of TEs may be functionally important during early 4 71 embryo development and understanding the regulation of TEs themselves is therefore of 72 great interest. 73 74 We adopted an unbiased approach to investigate the role of Stella during mouse maternal- 75 to-zygotic transition, using single-cell/embryo RNA-seq analysis of mutant embryos (Deng et 76 al. 2014, Xue et al. 2013, Yan et al. 2013). We find Stella M/Z KO 2-cell embryos fail to 77 upregulate key zygotic genes involved in regulation of ribosome and RNA processing. 78 Furthermore, the absence of Stella results in widespread misregulation of TEs and of 79 chimeric transcripts that are derived from these TEs. In particular, Stella M/Z KO embryos 80 exhibit a general failure to upregulate MuERV-L transcripts and protein at the 2-cell stage. 81 Our perturbation data is consistent with MuERV-L playing a functionally important role during 82 pre-implantation embryonic development, implying that MuERV-L is amongst the critical 83 factors affected by Stella in early embryos. 84 85 Results 86 87 Stella M/Z KO embryos are transcriptionally distinct from wild type embryos 88 We used Stella knockout mice (Payer et al. 2003), to collect mutant oocytes and embryos for 89 single-cell/embryo RNA-seq with modifications (Tang et al. 2010). Stella knockout (KO) 90 oocytes and Stella maternal/zygotic knockout (Stella M/Z KO, KO) 1-cell and 2-cell embryos, 91 lacking both maternal and zygotic Stella, were harvested. Strain matched wild type (WT) 92 oocytes and embryos served as controls (Figure 1A; Figure1 – Figure Supplement 2A). 93 Following normalisation, potentially confounding technical factors were controlled for using 94 Removal of Unwanted Variation (RUV) (Risso et al. 2014) (Figure 1 – Figure Supplement 2B, 95 Figure 1- source data 1, Supplementary File 1). 96 97 Principal component (PC) analysis revealed that the first PC represents progression from 98 oocyte to the 2-cell stage (Figure 1B). The largest separation is observed between oocyte / 5 99 1-cell and 2-cell embryos, consistent with the degradation of maternal transcripts and 100 activation of the zygotic genome at MZT. The second and third PCs capture the separation 101 between WT and KO samples, suggesting distinct genome-wide gene expression changes 102 between WT and KO embryos. Furthermore, although KO oocytes and 1-cell embryos 103 exhibit some separation from their WT counterparts, the difference is more clearly 104 pronounced at the 2-cell stage. Notably, Stella M/Z KO 1-cell and 2-cell embryos clustered 105 separately from each other, suggesting that Stella M/Z KO defects at the 2-cell stage are not 106 simply due to delayed developmental progression (Figure 1B). Differential gene expression 107 analysis between WT and KO samples identified 5881 misregulated genes (adjusted P- 108 value < 0.05) across the developmental stages with 360 genes overlapping across all stages 109 (Figure 1C, Figure 1 – source data 2). 110 111 Stella M/Z KO embryos are impaired in maternal-to-zygotic transition 112 Next, we compared the differentially expressed genes (DEG) against a public database of 113 early mouse embryonic transcriptomes (DBTMEE) (Park et al. 2015). This dataset 114 categorises genes by specific expression pattern at any developmental stage.